By developing the ability to follow single viruses as they assemble, it is possible to identify and characterize discrete steps in viral assembly. The earliest stages of this work established the criteria to be used in determining if a single virion, in this case of HIV-1, is being observed. This was followed by establishing criteria for determining and characterizing the packing of various components at the plasma membrane, for example the packing of Gag, the recruitment of the genome. This current project carries the work to the next level of identifying many more partial reactions in the process of viral assembly: When is the genome recruited? What is the interaction between the genome and Rev for packing, between the genome and Gag for packing? What determines the ability of the membrane to bend outward - is it just packing of Gag or are there additional factors? When are does the protease become active, what determines the apparent specificity of cleavage steps? When does the virion septate from the cell and, how do each of these steps inter-relate? Must the membrane bend to a certain degree to attain a sufficient proximity for the protease to activate or is it sufficient for Gag to pack to a critical spacing? What are the role(s) of the host ESCRT proteins? Do they simply facilitate budding or do they accelerate the assembly rate? With these assays of assembly at the level of the single virion, it becomes possible to final describe and define the process of assembly. PUBLIC HEALTH RELEVANCE: Viruses are a major threat to human health. This work has developed the ability to follow single viruses assembling in living cells. By elucidating the procedure of assembly, in this case for HIV-1, it opens the possibility of targeting these steps for disruption.